We seek to understand the molecular mechanisms of antioxidant protection in the mitochondria of eukaryotic cells. In particular, we plan to explore the relationships between the mitochondrial antioxidant enzymes, oxidative stress, and metal metabolism and to develop new tools to visualize cellular status in vivo. Our studies integrate the tools of inorganic chemistry, spectroscopy, and other biological methods with those of molecular biology, yeast genetics, and other biological methodology to elucidate in detail the chemical relationships between redox balance, oxidative stress, and metal ion metabolism in living cells. We will continue to use the budding yeast Saccharomyces cerevisiae as a model system for in vivo studies, and, in parallel, carry out related biochemical and biophysical studies using isolated antioxidant proteins. In this proposal, we follow several major lines of investigation focused on understanding the detailed mechanisms and the biological roles of proteins residing in the mitochondrial intermembrane space--copper- zinc superoxide dismutase (Sod1p), CCS (copper chaperone for Sod1p), and cytochrome c peroxidase. First, we propose a series of biological and genetic experiments designed to explore the roles of these proteins in protecting the mitochondrial matrix (as well as the intermembrane space) from oxidative damage. Included are studies on metabolic alterations that are beneficial for mutants that lack Sod1p involving a fourth IMS protein, lactate:cytochrome c oxidoreductase. In addition, we address the question of why the matrix enzyme manganese superoxide dismutase (MnSOD) does not fully protect the mitochondrial matrix under conditions of high superoxide flux. Second, we study the chemistry that allows ionic manganese functionally to substitute for Sod1p in yeast lacking this protein, including a potential role for cytochrome c peroxidase. Finally, we put forth and test a new hypothesis on the mechanism by which CCS activates Sod1, i.e., that the essential activity of CCS is formation of the disulfide bond in Sod1p, rather than insertion of copper as is commonly thought. In the course of these latter experiments, we will do a detailed characterization of the role of the disulfide bond in Sod1p. Together, these studies will shed light on the complex interactions of small molecules and proteins that all eukaryotes use to protect their mitochondria from superoxide-mediated damage.